Patient’s selection and tissue collection

The study was reviewed and approved by the Research Ethics Board of Mount Sinai Hospital, University of Toronto. We recruited white adult women undergoing vaginal hysterectomy for cervical prolapse equal or greater than stage III by POP-Q classification as “patients,” and women with stage 0 undergoing total abdominal hysterectomy for benign conditions other than POP as “controls.” We rationalized that stage 0 is the “gold standard” for normal pelvic support. Women with history of urogenital malignancy, endometriosis, connective tissue disorders, emphysema, previous pelvic surgery, and on estrogen and/or progestogen or steroid therapy were excluded. The initial gynecologic examinations were performed by the Urogynecology staff (H.P.D. and M.A.), and by the Gynecology team at Mount Sinai Hospital during regular activities. The first author obtained written informed consent, confirmed the POP staging of all participants, and collected clinical data a week before the surgical procedure. The patients were examined in the lying position with a referred full bladder, and asked to perform the Valsalva maneuver. The descensus of the vaginal compartments were measured at the maximum straining point using a centimeter scale ruler. Total vaginal length was measured at rest under POP reduction with a vaginal speculum. Afterward, straining examination in the standing position confirmed the full extent of the POP. We divided patients and controls in groups according to the menopausal status. We considered women in the postmenopausal phase if they reported that their menstrual periods had stopped for more than a year, and as premenopausal if they were having regular periods over the preceding 12 months. Only tissue samples from premenopausal women in the proliferative phase of the menstrual cycle were analyzed. The hormonal status was confirmed by endometrial histology report of uterine specimens. After removal of the uterus, vaginal tissue specimen (at least 1 cm ² ) was obtained by sharp dissection down to the avascular space of loose areolar connective tissue of the vagina using Metzenbaum scissors. The dissected structure corresponds to the adventitia layer that separates the vaginal from the bladder muscularis. As easily torn during dissection, adventitia was excluded from our analysis. To account for variations in stretch conditions and muscularis thickness throughout the vaginal length, the site of tissue collection was standardized at the anterior middle portion of the vaginal vault. The first author (M.B.), not blinded for the samples status, immediately received the tissue biopsies from the surgeon in the operative room and further performed the biochemical assays under direct supervision of the senior author (O.S.). For RNA and protein extraction, the vaginal samples were washed in ice-cold phosphate buffered saline solution (PBS), flash-frozen in liquid nitrogen and stored at −80°C. For immunohistochemical studies the specimens were sectioned longitudinally and fixed in 4% paraformaldehyde for 48 hours.

Real time-PCR analysis

Western immunoblot analysis

Tissues were crushed under liquid nitrogen, homogenized, and proteins were purified in RIPA lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1% (vol/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, supplemented with 100 μM sodium orthovanadate and protease inhibitor cocktail tablets (Complete Mini; Roche, Mississauga, Ontario, Canada). Protein concentrations were determined using the protein assay buffer (BioRad). Protein samples (40-60 μg) were solubilized in LDS sample buffer (Invitrogen, Carsbad, CA) and denatured by heating for 5 minutes. Lysates were resolved by electrophoresis on a gradient 4-12% Novex Tris-Glycine Pre-Cast Gels using Novex SDS Running Buffer (both Invitrogen). Protein extracted from human placenta was used as positive control, and brain tissue lysate (Abcam, Cambridge, MA) as negative control. We used PageRuler Plus Prestained Protein Ladder (SM1811; Fermentas, Burlington, CA) as molecular weight marker. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) using Novex Tris-Glycine Transfer Buffer 25× (Invitrogen). Blots were blocked with 5% skimmed milk/1% BSA, incubated with rabbit antihuman BMP1 primary antibody (1:3000; Abcam), washed in PBS-T and incubated with HRP-conjugated antirabbit IgG secondary antibody (1:3000; GE Healthcare, Chalfont, UK). Detection was performed using Immuno-Star Western C kit (Biorad). Membranes were stripped with Restore Plus Western Blot Stripping Buffer (ThermoScientific, Rockford, IL) and reprobed with housekeeping protein (ACTB) to correct variations in protein content among samples (rabbit antihuman ACTB primary antibody; 1:3000; BioVision, Mountain View, CA, and HRP-conjugated sheep antirabbit IgG secondary antibody; 1:3000; GE Healthcare). Probed membranes were exposed to Versadoc Imaging System 5000 MPs and quantified by densitometry using Quantity One Analysis Software (both Biorad). The values of relative optical density for POP patients were expressed in fold change relative to the corresponding controls.

Immunohistochemistry

The formalin fixed vaginal tissues were gradually dehydrated in ethanol and embedded in paraffin. Sections of 5 μm thickness were collected on Superfrost Plus slides (Fisher, Ontario, Canada). Paraffin sections were deparaffinized and rehydrated. After immersion in 3% hydrogen peroxide (Fisher Scientific, Fair Lawn, NJ), antigen retrieval was performed by treatment with 0.125% trypsin, followed by blocking with 5% normal horse serum and overnight incubation with primary antibody at 4°C, followed by appropriate secondary antibodies. To verify the morphology of the vaginal samples, we used alpha-smooth muscle actin immunostaining (rabbit antihuman ACTC1 primary antibody; 1:50; Dako, Glostrup, Denmark). For the BMP1 tissue localization, we used rabbit antihuman BMP1 primary antibody (1:1000; Abcam). BMP1 immunostaining of human umbilical cord tissue served as positive control. For the negative controls, ChromPure nonspecific rabbit IgG (Jackson Laboratories, West Grove, PA) was used in vaginal tissue at the same concentration as primary antibody. Biotin-conjugated goat antirabbit IgG (1:200; Vector Laboratories, Burlingame, CA) was used as secondary antibody. Counterstaining with Harris’ Hematoxylin (Sigma-Aldrich, St. Louis, MO) was carried out before slides were mounted with Cytoseal XYL (Ricard-Allan Scientific, Kalamazoo, MI). Vaginal tissue sections were observed on a DMRXE microscope (Leica Microsystems, Thornhill, Ontario, Canada). A minimum of 3 fields were examined for each specimen, and representative tissue sections were photographed with Sony DXC-970 MD 3CCD color video camera.

Statistical analysis

Pilot studies for quantification of BMP1 gene expression in premenopausal women indicated that 10 samples in each study group would be required to achieve a difference of at least 2-fold between women with and without POP at the P ≤ .05 for a power of 80%.

Unpaired comparisons between the expression of BMP1 gene and protein in POP patients and asymptomatic controls as well as premenopausal vs postmenopausal healthy women were performed using Wilcoxon signed-rank test (Prism version 4.02). Fisher’s exact test was used to compare demographic variables between the groups. The level of significance was set at P < .05. Experimental error was reported as SEM.